Studies of Energy Transport in Heart Cells. Mitochondrial Isoenzyme of Creatine Phosphokinase: Kinetic Properties and Regulatory Action of Mg2+ Ions

Abstract

1. The kinetic properties of mitochondrial creatine phosphokinase ( K , for all substrates and maximal rates of the forward and reverse reaction) have been studied. Since (a) K , value for MgADP-(0.05 mM) and creatine phosphate (0.5 mM) are significantly lower than K, for MgATP2-(0.7 mM) and creatine (5.0 mM) and (b) maximal rate of the reverse reaction (creatine phosphate + ADP -+ ATP + creatine) equal to 3.5 pmol x min-' x mg-' is essentially higher than maximal rate of the forward reaction (0.8 pmol x min-' x mg-'), ATP synthesis from ADP and creatine phosphate is kinetically preferable over the forward reaction.2. A possible regulatory role of Mg2+ ions in the creatine phosphokinase reaction has been tested. It has been shown that in the presence of all substrates and products of the reaction the ratio of the rates of forward and reverse reactions can be effectively regulated by the concentration of Mg2 + ions. At limited Mg2+ concentrations creatine phosphate is preferably synthesized while at high Mg2+ concentrations (more ATP in the reaction medium) ATP synthesis takes place.3 . The kinetic (mathematical) model of the mitochondrial creatine phosphokinase reaction has been developed. This model accounts for the existence of a variety of molecular forms of adenine nucleotides in solution and the formation of their complexes with magnesium. It is based on the assumption that the mitochondrial creatine phosphokinase reaction mechanism is analogous to that for soluble isoenzymes.4. The dependence of the overall rate of the creatine phosphokinase reaction on the concentration of total Mg2+ ions calculated from the kinetic model quantitatively correlates with the experimentally determined dependence through a wide range of substrates (ATP, ADP, creatine and creatine phosphate) concentration.The analysis of the kinetic model demonstrates that the observed regulatory effect of Mg2+ on the overall reaction rate can be explained by (a) the sigmoidal variation in the concentration of the MgADP-complex resulting from the competition between ATP and ADP for Mg2+ and (b) the high affinity of the enzyme to MgADP-.5. The results predicted by the model for the behaviour of mitochondrial creatine phosphokinase under conditions of oxidative phosphorylation point to an intimate functional interaction of mitochondrial creatine phosphokinase and ATP-ADP translocase.Myocardial cells are provided by energy largely due to the processes of aerobic oxidation of fatty acids and glucose and the synthesis of ATP in mitochondria [l, 21. The mechanism of oxidative phosAbbreviation.P. CP = creatine phosphate and Cr = creatine in equations; IU = international units of enzyme activity (pmol x min-I).Enzyme. Creatine phosphokinase or ATP : creatine phosphotransferase (EC 2.7.3.2).phorylation and the transport of adenine nucleotides across the inner mitochondrial membrane have been extensively elucidated in recent years [3 -61. However, further steps in the transport of energy from mitochondria to the active sites of its utilization on myofi...

Metabolic consequences of functional complexes of mitochondria,myofibrils and sarcoplasmic reticulum in muscle cells

“…An important observation shown in Table·1 is that activation of the mitochondrial creatine kinase (miCK) reaction decreases the value of apparent Km for exogenous ADP. This is due to functional coupling of the miCK reaction to the oxidative phosphorylation via the adenine nucleotide translocator (Barbour et al, 1984;Joubert et al, 2002;Saks et al, 1975Saks et al, , 1994Saks et al, , 1995Wallimann et al, 1992;Wyss and Kaddurah-Daouk, 2000), which leads to increased local turnover of adenine nucleotides in mitochondria, effective aerobic phosphocreatine production and metabolic stability of the heart (Garlid, 2001;Kay et al, 2000;Saks et al, 1995). This emphasizes the role of miCK in regulation of mitochondrial respiration in muscle cells (Joubert et al, 2002;Kay et al, 2000;Saks et al, 2001;Walsh et al, 2001).…”

“…For our calculations, dissociation constants of complexes of Mg 2+ with ADP and ATP were taken from Phillips et al (1966) and Saks et al (1975). 10·mmol·l -1 EGTA and 2.26·mmol·l -1 ATP were used as ligand concentrations, and 9.5·mmol·l -1 magnesium and 1.878·mmol·l -1 or 2.77·mmol·l -1 calcium were used for metals for calculations for solution A.…”

“…Mitochondria were isolated from the hearts of Wistar female rats as described previously (Saks et al, 1975).…”

The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability

“…Creatine phosphate then diffuses into the cytosol to buffer falling ATP levels. 51 The accumulation of creatine phosphate in mitochondria therefore suggests that transport across the OMM is impaired, while the continued production of creatine phosphate implies that ATP transport across the inner membrane is intact. 21 The defect in exchange across the OMM, with a subsequent stalling of the F 1 F 0 -ATPase's utilization of the hydrogen ion gradient, could also be responsible for the mitochondrial hyperpolarization that has been reported in response to multiple apoptotic stimuli.…”

Measurement of changes in high-energy phosphates in the cardiac cycle using gated 31P nuclear magnetic renonance.

“…The functional details of the energy reservoir system are far from certain, however. In one model (32)(33)(34), some or all of the high-energy phosphate produced by respiration in the mitochondria is exported to the rest of the cell as CrP. This CrP migrates to sites of utilization, such as myofibrils or sarcoplasmic reticulum, where it is converted by creatine phosphokinase to ATP.…”